Bombyx mori cytokine-like protein and application thereof

Abstract

The invention belongs to the technical field of biology, and provides application of bombyx mori cytokine-like protein as an active ingredient of a medicine for inhibiting bombyx mori nuclear polyhedrosis viruses, the amino acid sequence of the bombyx mori cytokine-like protein is shown as SEQ ID NO: 1, and the protein can inhibit replication of the bombyx mori nuclear polyhedrosis viruses. In addition, the invention also provides a bombyx mori cytokine-like protein, a bombyx mori cytokine-like plasmid is amplified through a PCR primer group to obtain a bombyx mori cytokine-like amplification product, then the bombyx mori cytokine-like amplification product and the bombyx mori cytokine-like plasmid are subjected to double enzyme digestion through incision enzyme, enzyme digestion products are purified and then connected through T4 ligase, and the bombyx mori cytokine-like protein is obtained. And transforming Escherichia coli DH5 alpha competent cells, screening positive clones, and extracting plasmids.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a silkworm cytokine-like protein and a use thereof. Background Art

[0002] Bombyx mori nucleopolyhedrovirus (BmNPV-eGFP) is a typical circular double-stranded DNA baculovirus. As an important viral pathogen of silkworm, the blood-type purulent disease caused by BmNPV-eGFP is the most common and most serious infectious disease in silkworm production. It is extremely harmful to the silkworm industry and causes serious economic losses to the silkworm industry every year. However, effective treatment for BmNPV-eGFP has not yet been found.

[0003] Chinese patent application 202410339412.1 discloses a method for expressing the protein of the antiviral gene Vago of Procambarus clarkii, comprising the following steps: cloning the antiviral gene Vago of Procambarus clarkii; constructing the recombinant plasmid pET28a-Vago using the prokaryotic expression vector pET28a, and then inducing expression using IPTG16 degrees; the target protein can be found in the supernatant, and the protein expression of the Vago gene of Procambarus clarkii is completed; the expressed purified vago protein and the white spot syndrome virus WSSV are injected into Procambarus clarkii respectively, and then cultured under a specific temperature environment; and blood samples of Procambarus clarkii are drawn to detect the expression changes of the main immune genes.

[0004] This method deeply explores the antiviral mechanism of Procambarus clarkii and uses the antiviral function of Vago to prevent and treat viral diseases of Procambarus clarkii. This program extracts the antiviral gene Vago from Procambarus clarkii and uses it to prevent and treat viral diseases of Procambarus clarkii.

[0005] Chinese patent application 202311457695.1 discloses the use of glutathione in the preparation of a drug for preventing or treating Bombyx mori nuclear polyhedrosis virus disease. By using a drug containing glutathione at an effective concentration of 5mM to 10mM, glutathione against Bombyx mori nuclear polyhedrosis virus is rationally utilized to inhibit its replication in cells. When infection occurs, the drug can be mixed with the silkworm's food and eaten to exert a therapeutic effect. Alternatively, glutathione can be added to the food 24 hours before virus infection to inhibit the proliferation of BmNPV-eGFP and prevent infection.

[0006] This method shows that the silkworm peptide glutathione can effectively inhibit the replication of Bombyx mori nuclear polyhedrosis virus (BmNP V-eGFP) in silkworm cells. Therefore, glutathione can be used to inhibit the replication of the virus in silkworm cells and silkworms through appropriate concentrations and drug addition time.

[0007] The problem that this project needs to solve is: How to propose a new approach to inhibit the replication of Bombyx mori nuclear polyhedrosis virus in Bombyx mori. Summary of the invention

[0008] The object of the present invention is to provide a silkworm cytokine-like protein which can effectively inhibit the replication of Bombyx mori nuclear polyhedrosis virus in Bombyx mori, and the protein inhibits the replication of Bombyx mori nuclear polyhedrosis virus through the JAK-STAT signaling pathway.

[0009] To achieve the above object, the present application discloses the use of a silkworm cytokine-like protein as an active ingredient of a drug for inhibiting the silkworm nuclear polyhedrosis virus.

[0010] Preferably, the amino acid sequence of the silkworm cytokine-like protein is as shown in SEQ ID NO: 1;

[0011] The encoding nucleotide sequence of the silkworm cytokine-like protein is shown in SEQ ID NO:2.

[0012] Preferably, the silkworm cytokine-like protein inhibits the replication of Bombyx mori nuclear polyhedrosis virus via the JAK-STAT signaling pathway.

[0013] In addition, a silkworm cytokine-like protein is disclosed. The silkworm cytokine-like protein is obtained by amplifying a silkworm cytokine-like plasmid with a PCR primer set to obtain a silkworm cytokine-like amplification product, and then double-digesting the silkworm cytokine-like amplification product and the silkworm cytokine-like plasmid with an endonuclease, purifying the digestion products, connecting them with T4 ligase, transforming Escherichia coli DH5α competent cells, screening positive clones, and extracting plasmids.

[0014] Preferably, the PCR primer set comprises an upstream primer F1 and a downstream primer R1;

[0015] The sequence of the upstream primer F1 is shown in SEQ ID NO: 3;

[0016] The sequence of the downstream primer R1 is shown in SEQ ID NO:4.

[0017] Preferably, the endonucleases are respectively the nuclease BamHI enzyme for the Bombyx mori cytokine-like amplification product and the restriction endonuclease SacI enzyme for the Bombyx mori cytokine-like plasmid.

[0018] Beneficial effects of this application:

[0019] The present application provides a silkworm cytokine-like protein that can effectively inhibit the replication of Bombyx mori nuclear polyhedrosis virus in Bombyx mori. The protein inhibits the replication of Bombyx mori nuclear polyhedrosis virus through the JAK-STAT signaling pathway, and the protein can also provide a theoretical reference for the development of anti-BmNPV-eGFP drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of silkworm cytokine expression in BmN cells after BmNPV-eGFP infection for 24h, 48h, and 72h.

[0021] Figure 2 The results are for the expression of silkworm cytokines 72 hours after BmNPV-eGFP infection in silkworm tissues;

[0022] Figure 3 The signal peptide prediction results of proteins using the NovoPro website;

[0023] Figure 4 The results of Western Blot detection of silkworm cytokine-like proteins, where the left band is the result of cytokine expression detected in the culture supernatant; the right band is the result of cytokine expression detected in the cell pellet;

[0024] Figure 5 The overexpression vector BmLOC101736094 was transfected into BmN cells and then infected with BmNPV-eGF P. The expression level of vp39 was detected after 36h and 48h.

[0025] Figure 6 The results of protein expression detection by SDS-PAGE, where M is protein marker, 1 and 2 are empty vectors, ①②③ represent different positive strains, 3, 7, 11 are uninduced bacterial solutions, 4, 8, 12 are induced bacterial solutions, 5, 9, 13 are induced bacterial solution precipitates, and 6, 10, 14 are induced bacterial solution supernatants;

[0026] Figure 7 The results of protein purification by SDS-PAGE, where the lanes are: M is protein marker, 1 is bacterial stock solution, 2 is the supernatant after bacterial disruption, FT1 is 20mM imidazole washing solution, FT2 is 50mM imidazole washing solution, FT3 is 100mM imidazole washing solution, E1~E6 are 250mM imidazole elution solution;

[0027] Figure 8 The results of Western Blot verification of purified protein, where the lanes are: M is protein marker, 1 is protein solution before renaturation, 2 is protein solution after renaturation;

[0028] Fig. 9 The cytotoxicity results after BmN cells were treated with B. mori cytokine-like protein at concentrations of 0.6 μg / mL, 1.8 μg / mL, and 3 μg / mL;

[0029] Fig.10 The expression results of vp39 at 48h and 72h after adding BmNPV-eGFP to the knockout cell line KO-STAT;

[0030] Fig.11 The results of the inhibition of the replication of BmNPV-eGFP gene vp39 by 0.6 μg / mL of Bombyx mori cytokine-like protein;

[0031] Fig.12 The expression results of BmNPV-eGFP gene vp39 by adding silkworm cytokine-like protein to the knockout cell line KO-STAT. DETAILED DESCRIPTION

[0032] In the description of this application, it should be noted that if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0033] The sources of reagents are shown in Table 1:

[0034] Table 1

[0035]

[0036]

[0037] Example 1

[0038] Changes in the level of silkworm cytokine-like transcription in BmN cells after BmNPV-eGFP infection. The specific experimental process is as follows:

[0039] (1) Bombyx mori ovary cells (BmN) were inoculated into 12-well cell culture plates and cultured in a 28°C incubator overnight. BmNPV-eGFP recombinant fluorescent reporter virus (multiplicity of infection: 5) was added to the culture plate, incubated at 28°C for 1 hour, and then replaced with fresh Grace medium containing 10% FBS. Cell samples were collected at 24h, 48h, and 72h after infection.

[0040] (2) 5 μL of BmNPV-eGFP recombinant fluorescent reporter virus was injected into the fifth-instar "Da Zao" silkworm larvae by tail-foot injection. Silkworm tissue samples were collected 3 days after infection: fat body, midgut, hemocytes, head, silk gland, Malpighian tubules, trachea, and epidermis.

[0041] (3) 100 μL of Trizol lysis solution (RNA extraction reagent) was added to the harvested cells, and 1 mL of Trizol solution was added to the silkworm tissue for homogenization. Total RNA was extracted from cells and tissues using Kit RNA fast 2000 (RNA extraction kit) according to the manufacturer's instructions, and RNA was reverse transcribed into cDNA using the gDNA Eraser (reverse transcription kit) kit.

[0042] (4) Using TIF4A as an internal reference, the transcriptional level of cytokine-like gene in silkworms infected with BmNPV-eGFP was detected by real-time fluorescence quantitative PCR. The PCR reaction system was configured according to the instructions of Green Supermix Kit reagents. qPCR detection was performed using a fluorescent quantitative PCR instrument (CFXTM Optics Module, Bio-Rad, USA).

[0043] The results are as follows Figure 1 As shown in Figure 2, the expression level of the silkworm cytokine BmLOC101736094 in silkworm cells increased significantly at an exponential rate at 24hpi, 48hpi, and 72hpi after infection with the BmNPV-eGFP virus. Figure 2 It can be seen that the expression level of silkworm cytokine BmLOC101736094 was most significantly upregulated in the three major silkworm larvae tissues: midgut, hemocytes and Malpighian tubules.

[0044] Example 2

[0045] It is proved that BmLOC101736094 is a secreted protein and can play an antiviral role at the protein level

[0046] (1) Bombyx mori ovary cells (BmN) were inoculated into 12-well cell culture plates and cultured in a 28°C incubator overnight. The constructed overexpression vector pIEX-BmLOC101736094 was transfected into BmN cells at a concentration of 1000 ng per well, and Western Blot cell samples were collected 24 h, 48 h, 72 h, and 96 h after transfection.

[0047] (2) Add 80ul of RIPA cell lysis buffer and 2ul of PMSF protease inhibitor to the harvested cells. After ice bathing for 30min, add 20ul of 5× loading buffer to prepare protein samples for subsequent Western Blot.

[0048] (3) Similarly, Bombyx mori ovary cells (BmN) were inoculated into 12-well cell culture plates and cultured in a 28°C incubator overnight. The constructed overexpression vector pIEX-BmLOC101736094 was transfected into BmN cells at a concentration of 1000 ng per well, and 1 MOI of BmNPV-eGFP virus was added for infection 24 hours later. RNA cell samples were collected at 36 hours and 48 hours later.

[0049] (4) 100 μL of Trizol lysis solution (RNA extraction reagent) was added to the harvested cells, and 1 mL of Trizol solution was added to the silkworm tissue for homogenization. Total RNA was extracted from cells and tissues using Kit RNA fast 2000 (RNA extraction kit) according to the manufacturer's instructions, and RNA was reverse transcribed into cDNA using the gDNA Eraser (reverse transcription kit) kit.

[0050] (5) Using TIF4A as an internal reference, the transcriptional level of cytokine-like gene in silkworms infected with BmNPV-eGFP was detected by real-time fluorescence quantitative PCR. The PCR reaction system was configured according to the instructions of Green Supermix Kit reagents. qPCR detection was performed using a fluorescent quantitative PCR instrument (CFXTM Optics Module, Bio-Rad, USA).

[0051] The results are as follows Figure 3 As shown, BmLOC101736094 contains a 17 amino acid signal sequence at the 5' end, indicating that BmLOC101736094 is a secreted protein containing a signal peptide.

[0052] like Figure 4 As shown, after the constructed overexpression vector pIEX-BmLOC101736094-his was transfected into the cells, the cell internal reference antibody GAPDH was used for detection. A clear and bright band could be detected from the cell precipitate, but no band was detected in the culture supernatant, which proved that the sample was the cell culture supernatant; then the tag antibody anti-his was used to detect the expression of the target protein in both the cell culture supernatant and the precipitate. It can be seen that the cell culture supernatant sample is correct and the expression of the target protein BmL OC101736094 can be detected, thus indicating that the silkworm cytokine BmLOC101736094 is a type of secreted protein;

[0053] like Figure 5As shown, when the silkworm ovarian cells transfected with BmLOC101736094 secretory protein were infected with BmNPV-eGFP virus, compared with the control group, at 36hpi after virus infection, the expression level of vp39 in the BmNPV-e GFP virus dropped from 1.0 to around 0.6, showing a significant change. It can be seen that the BmLOC101736094 secretory protein can inhibit the replication of the BmNPV-eGFP virus, and when further observed at 48hpi, the expression level of vp39 in the BmNPV-eGFP virus can also drop from 0.6 to around 0.5, and the inhibitory effect of the BmLOC101736094 secretory protein can be further enhanced.

[0054] Example 3

[0055] Codon Optimization of the Silkworm Cytokine-like Gene and Construction of the Expression Vector

[0056] The open reading frame sequence of silkworm cytokine-like gene (Gene ID: 101736094) was obtained from NCBI. The sequence was first analyzed by bioinformatics. The sequence contained a 17-amino acid signal peptide at the 5' end. The gene sequence was codon optimized, and a BamⅠ restriction site was added to the 5' end of the sequence, a SacⅠ restriction site was added to the 3' end, the signal peptide sequence MTMLAFVLVGLITTISA was removed, and a 6*H is tag was added to the C-terminus to obtain the codon-optimized silkworm cytokine-like gene. Its amino acid sequence is as follows: MT MLAFVLVGLITTISAGPSNDSFKDQYCTDPRTMQKHAAYSEWADAYSCTRHRCQPGGRNLAIYTVGCKRVEAPESAIECEEVVEDTNMQFPFCCTRLRCLVVVRGEVWTRVLGQPWETLPAAPWS HMYKMKKPPPGDTSFLNKETEQKGPVYELSEQDAPKHEQVLRSEKRTTEDPNCKEAVLRSAPTPDIVIALSTNNDEKEPKRDIERRKRVHEIPDEERNEPENQDVEELPASTDKPLGNSDEAYNS NTDAMTKKPKTKIQNQVTWTEIPPNQWTERDPSMDAENVKDVQEEKKEVKSSNLQALVDAIGTRMRDIETVVQKMSQKVQQVKPEDAVASSEKRSDEGKRHERKEHEKNYDELKKPSPDGDHYKR FVVSSHSKYLHSADGVTEKPLYIEDNTNGYYSDASNSVIRRVNVPKEPSPTYMAPVETRRKSIHPVPENSEEVGKKRKKHSHKKKGKGKSHKKHRKDEKRRRYNRISSEVDKNVVSLEDNSANKK.

[0057]

[0058] The synthesized codon-optimized silkworm cytokine-like gene sequence was used as a template, and the primers in Table 2 were used for PCR amplification to obtain the silkworm cytokine-like fragment. The amplified product was purified and recovered, and the amplified product and the pET-28a plasmid were double-digested with Bam HI enzyme and S ac I enzyme, respectively. After the digestion products were purified, they were ligated with T4 ligase, transformed into Escherichia coli DH5α competent cells, positive clones were screened, plasmids were extracted, and enzyme digestion verification was performed. The recombinant vector verified by enzyme digestion was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. Guangzhou Branch for sequencing confirmation, and the recombinant plasmid pET-28a-BmLOC101736094 silkworm cytokine-like was obtained.

[0059] Table 2

[0060]

[0061] Example 4

[0062] Expression and purification of BmLOC101736094 silkworm cytokine-like

[0063] The successfully constructed silkworm cytokine-like recombinant plasmid pET-28a-BmLOC101736094 was transformed into Escherichia coli Rosetta (DE3) competent cells by heat shock method, and positive clones were screened by kanamycin and chloramphenicol resistance plates, and double enzyme digestion was used to further verify the prokaryotic expression recombinant positive plasmid.

[0064] The silkworm cytokine-like Rosetta (DE3) E. coli / pET-28a-BmLOC101736094 identified again was inoculated into LB liquid culture medium containing kanamycin and cultured at 37°C with shaking. When the OD 600 value of the culture reached 0.6, IPTG with a final concentration of 0.1 mmol / L was added. After continued induction at 32°C for 8 h, the bacteria were collected by centrifugation at 8000 g / min for 5 min, resuspended in 1:10 volume of PBS, and ultrasonically disrupted. The supernatant / precipitate was collected and detected by SDS-PAGE.

[0065] from Figure 6 It can be seen that compared with the empty load group, protein content was detected in the whole bacteria, precipitate and supernatant of the three different positive strains; and the protein expression levels in lanes 5, 9 and 13 were significantly higher than those in lanes 6, 10 and 14, that is, the protein content of the strain precipitate was higher than that of the supernatant.

[0066] Example 5

[0067] Expression product extraction and protein refolding and purification

[0068] (1) Extraction and processing of inclusion bodies:

[0069] 500 ml of the induced expression recombinant bacteria E.coli / pET-28a-BmLOC101736094 was centrifuged at 8000g / min for 10 min to collect the bacteria, and the bacteria were resuspended in an ultrasonic buffer at a ratio of 1:10, and fully ultrasonically disrupted on ice, wherein the ultrasonic buffer was 100mM NaH2PO4, 300mM NaCl, 100mM Tris, pH7.2; then, the precipitate was collected by centrifugation at 4°C and 12000g / min for 20 min, and the precipitate was the crude inclusion body.

[0070] The crude inclusion bodies collected by centrifugation were magnetically stirred at room temperature for 1 h with a washing buffer consisting of 100 mM NaH2PO4, 300 mM NaCl, 100 mM Tris, 2% NP-40, pH 7.2, followed by centrifugation at 4°C and 12,000 g / min for 20 min.

[0071] Then the inclusion bodies were washed with washing solution 2 and washing solution 3 in turn; the washing solution 2 was 100mM NaH2PO4, 300mM NaCl, 100mM Tris, 2M Uera, pH7.2; the washing solution 3 was 100mM NaH2PO4, 300mM NaCl, 100mM Tris, 4M Uera, pH7.2.

[0072] (2) Denaturation of inclusion bodies

[0073] The inclusion body denaturation solution was added to the washed inclusion bodies at a ratio of 1:10. The denaturation solution was 100mM NaH2PO4, 300mM NaCl, 100mM Tris, 8M Uera, pH7.2. The mixture was fully stirred by magnetic force at room temperature for 2h. The supernatant was collected by centrifugation at 4°C and 12000g / min for 30min. The supernatant was filtered with a 0.45mm filter membrane and stored at 4°C.

[0074] (3) Purification of inclusion bodies

[0075] The His-tagged supernatant was purified according to the nickel column purification instructions of Solarbio, and then the purified product was analyzed by SDS-PAGE.

[0076] The results are as follows Figure 7 , Figure 8 As shown, Figure 7The results of protein purification by SDS-PAGE, lane 1 is the expression of the target protein detected in the stock solution, lane 2 is the flow-through after the stock solution containing the target protein is combined with the nickel column, lanes FT1, FT2, and FT3 are elution solutions containing 20mM, 50mM, and 100mM imidazole, respectively, which can remove some non-target proteins, and then the target protein eluted from the nickel column is detected from the eluents of lanes E1, E2, E3, E4, and E5; Figure 8 Western Blot was used to verify the purified protein results. The results showed that the SDS-PAGE purified protein had a single electrophoresis band.

[0077] Example 6

[0078] (1) Cytotoxicity assay

[0079] According to the instructions of the Cell Counting Kit-8 (purchased from Bio-Technology Co., Ltd.), BmN cells were seeded in a 96-well plate at a cell density of 1×10 4 BmN cells were treated with B. mori cytokine-like protein at concentrations of 0.6 μg / mL, 1.8 μg / mL, and 3 μg / mL for 24 h, and the culture medium in each well was replaced with a culture medium containing CCK-8 (10 μL per well), and culture was continued for 4 h. The OD450nm value was detected using an ELISA reader.

[0080] Depend on Fig. 9 It can be seen that the increase in protein dosage has no significant effect on cell growth activity. According to the principle of minimum protein dosage, 0.6ug / ml is determined to be the optimal protein concentration.

[0081] (2) Cell antiviral assay

[0082] After infection with 1 MOI of BmNPV-eGFP in the KO-STAT cell line, the supernatant was replaced with Grace medium (10% FBS) 1 hour later. Cell samples were collected 48h and 72h after virus infection. Total RNA was extracted according to the instructions of the Flytek Total RNA Rapid Extraction Kit, and the expression changes of the viral vp39 gene were detected using real-time fluorescence quantitative PCR.

[0083] Fig.10 The results showed that after STA T, the key gene of the Jak-STAT signaling pathway, was knocked out in the KO-STAT cell line, the expression level of the BmNPV-eGFP virus vp39 gene in the KO-STAT cell line was significantly upregulated. This shows that the JAK-STAT signaling pathway plays an important role in inhibiting the replication of the BmNPV-eGFP virus.

[0084] (3) Silkworm cytokine-like protein exerts antiviral function through the JAK-STAT signaling pathway

[0085] Then, the BmN cells were treated with 0.6 μg / mL of silkworm cytokine-like protein for 24 hours, and the BmN cells were infected with 1 MOI of BmNPV-eGFP. PBS was used as the control group. After 1 hour, the supernatant was replaced with Grace medium (10% FBS). Cell samples were collected 24 hours, 48 ​​hours, and 72 hours after virus infection. According to the instructions of the Fijet Total RNA Rapid Extraction Kit, total RNA was extracted, and the expression changes of the viral vp39 gene were detected by real-time fluorescence quantitative PCR.

[0086] Fig.11 The results showed that after adding silkworm cytokine-like protein to BmN cells, real-time fluorescence quantitative PCR detection showed that compared with the control group, the expression level of vp39 gene in BmNP V-eGFP virus in BmN cells transfected with silkworm cytokine-like protein showed a significant decrease at 24hpi, 48hpi and 72hpi after infection with BmNPV-eGFP virus. Fig.11 It can be seen that the inhibitory effect of silkworm cytokine protein on BmNPV-e GFP virus is stronger at 24hpi after BmNPV-eGFP virus infection, decreasing from 1.0 to about 0.8, a decrease of 0.2. At 48hpi and 72hpi, it only decreased to 0.7 and 0.6 respectively, a decrease of 0.1.

[0087] In summary, Bombyx mori cytokine-like protein can effectively inhibit the replication of BmNPV-eG FP virus in BmN cells.

[0088] At the same time, the KO-STAT cell line was treated with 0.6 μg / mL of silkworm cytokine-like protein for 24 hours, and BmN cells were infected with 1 MOI of BmNPV-eGFP. PBS was used as the control group. After 1 hour, the supernatant was replaced with Grace medium (10% FBS). Cell samples were collected 48 hours and 72 hours after virus infection. According to the instructions of the Fijet Total RNA Rapid Extraction Kit, total RNA was extracted, and real-time fluorescence quantitative PCR was used to detect the expression changes of the viral vp39 gene.

[0089] Fig.12 The results showed that after adding silkworm cytokine-like protein to the KO-STAT cell line, real-time fluorescence quantitative PCR detection found that the expression level of vp39 gene in the BmNPV-eGFP virus was significantly increased at 48hpi. At the same time, after observation at 72hpi, comparison with the control group also found that the expression level of vp39 gene in the BmNPV-eGFP virus still showed a significant increase.

[0090] And further observation Fig.11 , Fig.12 It can be seen that in normal BmN cells, BmLOC101736094 protein can inhibit the replication of BmNPV-eGFP virus, while in KO-ST AT cells with JAK-STAT signaling pathway knocked out, BmLOC101736094 protein cannot inhibit the proliferation of the virus. It can be seen that the silkworm cytokine protein BmLOC101736094 inhibits the infection of BmNPV-eGFP through the JAK-STAT signaling pathway.

[0091] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of silkworm cytokine-like protein as an active ingredient for inhibiting silkworm nuclear polyhedrosis virus.

2. The use according to claim 1, characterized in that The amino acid sequence of the silkworm cytokine-like protein is shown in SEQ ID NO: 1; The encoding nucleotide sequence of the silkworm cytokine-like protein is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that The silkworm cytokine-like protein inhibits the replication of the silkworm nuclear polyhedrosis virus through the JAK-STAT signal pathway.

4. A silkworm cytokine-like protein, characterized in that The silkworm cytokine-like protein is amplified by a PCR primer set on a silkworm cytokine-like plasmid to obtain a silkworm cytokine-like amplified product, and then the silkworm cytokine-like amplified product and the silkworm cytokine-like plasmid are double-digested by endonucleases, the digested products are purified and connected by T4 ligase, and transformed into Escherichia coli DH5α competent cells, positive clones are screened to obtain a recombinant plasmid, and then the recombinant plasmid is transfected into Escherichia coli Rosetta competent cells for expression to obtain the silkworm cytokine-like protein.

5. The silkworm cytokine-like protein according to claim 4, characterized in that The PCR primer set includes an upstream primer F1 and a downstream primer R1; The sequence of the upstream primer F1 is shown in SEQ ID NO: 3; The sequence of the downstream primer R1 is shown in SEQ ID NO:

4.

6. The silkworm cytokine-like protein according to claim 4, characterized in that The silkworm cytokine-like amplified product and the silkworm cytokine-like plasmid were double-digested with Bam HI enzyme and Sac I enzyme respectively.

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